The ecological risks of antibiotics in aquatic environments have raised great concerns worldwide, but the chronic effect of antibiotic contaminants on cyanotoxin production and release remains unclear. This study investigated the long-term combined effects of spiramycin (SP) and ampicillin (AMP) on microcystin (MC) production and release in both unicellular and colonial Microcystis aeruginosa (MA) through semi-continuous exposure test. At exposure concentration of 300 ng L, MA growth rates were stimulated till the end of exponential phase accompanied with the up-regulation of photosynthesis-related gene. The exponential growth phases of unicellular and colonial MA were prolonged for 2 and 4 days, respectively. The stimulation rate of growth rate and MC content in unicellular MA were significantly higher than that in colonial MA. The highest concentrations of intracellular MC (IMC) and extracellular MC (EMC) were observed in the binary mixture at equivalent SP/AMP ratio (1:1). The promotion of IMC concentration was in consistent with the stimulated expression of MC-synthesis-related gene and nitrogen-transport-related gene. The malondialdehyde content and activities of superoxide dismutase and catalase in unicellular MA were significantly higher than those in colonial MA. The EMC concentration and the antioxidant responses of both unicellular and colonial MA significantly increased with exposure time. Long-term exposure to mixture of SA and AMP at environmentally relevant concentrations would aggravate the disturbance to aquatic ecosystem balance through the stimulation of MA proliferation as well as the promotion of MC production and release.