Abstract: Objective　To investigate the antimicrobial activity of omadacycline (OMC) against clinical Streptococcus agalactiae (GBS) isolates, as well as its relationship with biofilm formation, resistance genes and virulence genes. Methods　A total of 136 strains of Streptococcus agalactiae isolated from Shenzhen Nanshan People's Hospital between 2015 to 2020. The minimum inhibitory concentration (MIC) of OMC against Streptococcus agalactiae was determined by broth microdilution. Crystal violet staining was used to detect the biofilm formation ability of GBS. Resistance genes (tetM, tetO, tetK, ermB, OptrA) and virulence genes (cpsⅢ, bca, fbsA, cpsA, scpB) were investigated by polymerase chain reaction (PCR). Results　Among the 136 clinical isolates of GBS, 20 strains (14.7%) were resistant to OMC, 64 (47.1%) were intermediate, and 52 (38.2%) were sensitive. Fifty-seven strains (41.9%) were biofilm-positive, 20 of which (35.1%) were sensitive to OMC. Seventy-nine strains (58.1%) were biofilm-negative, 32 of which (40.5%) were susceptible to OMC. There was a statistically significant difference in the sensitivity rates between the two groups of strains (χ2=63.062, P<0.001), but there was no significant difference in the sensitivity of OMC among the biofilm-positive strains (Fisher's exact test, P=0.824). The resistance rates of tetM, tetO, ermB and OptrA positive strains were higher than those of negative strains, while tetK was opposite. The presence of tetM (Z=0.815, P=0.415), tetO (Z=0.151, P=0.88), tetK (Z=0.567, P=0.571), ermB (Z=1.198, P=0.231) resistance genes in Streptococcus agalactiae had no significant impact on the sensitivity of OMC. However, the presence of the OptrA resistance gene showed a statistically significant effect on the sensitivity of OMC (Z=2.913, P=0.004). The virulence factors cpsⅢ, bca, fbsA, cpsA and scpB were all detected at a rate higher than 50%. The presence of the virulence genes cpsⅢ (Z=0.222, P=0.824), bca (Z=0.141, P=0.888), fbsA (Z=0.813, P=0.416), and cpsA (Z=1.615, P=0.106) in Streptococcus agalactiae had no significant impact on the sensitivity of OMC. However, there was a significant inter-group difference in the scpB virulence gene (Z=2.844, P=0.004), but the rank mean values and resistance rates of scpB-positive strains were lower than those of the negative strains. Conclusions　The formation of biofilm in Streptococcus agalactiae reduces its sensitivity to OMC, but there was no significant difference in the sensitivity to OMC among the biofilm-positive strains. The presence of resistance genes tetM, tetO, tetK, ermB, and virulence genes cpsⅢ, bca, fbsA, cpsA, scpB in Streptococcus agalactiae is not associated with OMC resistance, but the presence of the resistance gene OptrA is correlated with OMC resistance..