Regarding increasing rate of drug resistance among microbial pathogens, a global search to find new antimicrobial agents from natural compounds with fewer side effects has been considered by many researchers worldwide. Bioactive compounds with good antimicrobial activity have been isolated from different algae and cyanobacteria. The current study was performed to determine antimicrobial potential and to characterize the effect of algal growth modes (autotrophic, heterotrophic and mixotrophic) and extraction solvents on antimicrobial activity of the microalgae Chlorella vulgaris against four bacterial and one fungal pathogens. C. vulgaris was grown under different growth conditions and the biomass was harvested. Different extracts were prepared using acetone, chloroform and ethyl acetate as extraction solvents and antimicrobial activity of the extracts was investigated against two Gram positive bacteria (Bacillus subtilis and Staphylococcus aureus), two Gram negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and a fungal strain (Candida albicans) using agar well diffusion assay. In addition, Minimal Inhibitory Concentration (MIC) of the extracts were determined. Moreover, the total oil content and lipid productivity of C. vulgaris grown under different modes were determined. The heterotrophic growth resulted in stronger antimicrobial activity compared to the other growth conditions. In addition, the highest antimicrobial activity was observed for chloroform mediated extract and extraction using acetone resulted in minimum antimicrobial activity. Moreover, heterotrophic and mixotrophic growth significantly increased the total lipid content and lipid productivity compared to the autotrophic growth. C. vulgaris exhibited good antimicrobial potential and the antimicrobial efficacy could be influenced by extraction solvents and growth conditions.