The green algaChlorella vulgarishas potential as a source of clean bioenergy with abundant metabolites and a high oil content, and antibiotics are often applied to remove bacteria from culture to obtain axenic algal strains. In this work, ceftazidime and gentamicin sulphate (GS) were added separately and in combination in the aseptic processing ofC. vulgaris, and gene expression and metabolites were evaluated. The results showed that ceftazidime and GS effectively inhibited the proliferation ofCyanobacteriaandBacteroidetes, respectively. Overall, the effects of antibiotics onC. vulgarisdiffered: GS increased the algal concentration, whereas ceftazidime alone and in combination with GS treatment decreased the specific algal growth rate. Based on comparative transcription analysis, 5917 and 5899 differentially expressed genes (DEGs) were respectively upregulated and downregulated by ceftazidime, 963 and 3921 DEGs by GS, and 4532 and 1675 DEGs by the ceftazidime and GS combination. Pathway enrichment analysis showed that the downregulated DEGs in the ceftazidime groups were enriched in the fatty acid biosynthesis pathway but that the upregulated DEGs in the GS group were enriched in the fatty acid degradation pathway. Some pathways related to amino acid metabolism were markably influenced by antibiotic treatment. The results further indicated that antibiotics affected the intracellular concentration of fatty acids and amino acids inC. vulgaris. This study provides a new viewpoint regarding the response ofC. vulgaristo antibiotics in the process of obtaining axenic algal strains.