Chaetoceros muelleri and Isochrysis zhanjiangensis, known for their rapid reproduction, small size, and rich nutritional content, are commonly used as feed microalgae in aquaculture. This study aimed to sterilize these microalgal species and assess the effects of antibiotics on their algal cell density. Phycospheric bacteria were isolated and identified using the spread plate method and 16S rDNA sequencing, and antibiotic susceptibility tests were conducted using four antibiotics: ampicillin, streptomycin, kanamycin, and gentamicin sulfate. A sterile system was established for C. muelleri using ampicillin, streptomycin, and gentamicin, and for I. zhanjiangensis using kanamycin, ampicillin, and streptomycin. Based on the results, antibiotics with sterilization effects were selected and added to the algal cultures. Their effects on cell density were evaluated during a six-day co-culture. Ampicillin and streptomycin effectively inhibited bacteria associated with C. muelleri, initially increasing algal cell density but later causing a decline. For I. zhanjiangensis, kanamycin and ampicillin were effective, with kanamycin significantly promoting growth throughout the cycle, achieving a 36.92% higher cell density on day six (p < 0.05).