Objective Prototheca is a unicellular microalga belonging to the family Chlorellaceae that has lost its chlorophyll and the ability to perform photosynthesis, exhibiting biological characteristics intermediate between algae and fungi. Protothecosis is predominantly an exogenous infection, with pathogens commonly invading through damaged skin or mucous membranes via contact with contaminated soil, water, or organic matter. Clinical manifestations primarily include cutaneous and subcutaneous infections and olecranon bursitis. This study investigates the epidemiological characteristics and antimicrobial susceptibility distribution of protothecosis in China from 2015 to 2025, aiming to bridge the gap in domestic epidemiological data and clinical management guidelines of protothecosis, and to provide a scientific basis for its clinical diagnosis, treatment, and prevention strategies. Methods A total of 84 clinical Prototheca isolates collected by the Protothecosis Science Popularization and Monitoring Consortium (PSPMC) between 2015 and 2025 were retrospectively analyzed. In vitro susceptibility testing was performed for all isolates using the broth microdilution method. The tested agents included five azoles, three echinocandins, 5-flucytosine, and amphotericin B. Additionally, CYTB gene sequencing was conducted, followed by phylogenetic analysis. Results Among the isolates obtained from patients with protothecosis in China between 2015 and 2025, Prototheca wickerhamii was the predominant species (79.8%), followed by Prototheca bovis (11.9%). The highest number of isolates was found in East China (45.2%). The infections predominantly affected middle-aged and elderly individuals, with 82.1% of patients aged between 41 and 80 years. Skin tissue was the most common isolation source, accounting for 59.5% of all clinical isolates. Non-cutaneous sources included cerebrospinal fluid, blood, ascitic fluid, and bronchoalveolar lavage fluid. Azole antifungal agents, such as itraconazole, posaconazole, and voriconazole, demonstrated strong antimicrobial activity against Prototheca, with the minimum inhibitory concentration (MIC) required to inhibit 50% of isolates (MIC₅₀) being 1.000 μg/mL. Isavuconazole exhibited superior activity, with MICs ranging from 0.015 to 1.000 μg/mL. Amphotericin B also showed strong antimicrobial activity (MIC₅₀ = 0.500 μg/mL). Conversely, fluconazole, 5-flucytosine, and echinocandins showed no significant antimicrobial activity against Prototheca. The antimicrobial activity of isavuconazole against P. wickerhamii was significantly higher than against P. bovis and Prototheca zopfii. Phylogenetic analysis indicated that P. wickerhamii constituted the major evolutionary clade with a highly complex structure, suggesting significant intraspecific genetic heterogeneity. Conclusions Prototheca wickerhamii is the predominant species among isolates obtained from patients with protothecosis in China. The infected population mainly consists of middle-aged and elderly individuals. While skin is the main site of infection, non-cutaneous sites are widely distributed. Amphotericin B and isavuconazole demonstrate strong in vitro antimicrobial activity against these clinical Prototheca isolates, although susceptibility varies among different species.