Surgical site infection (SSI) is a common complication that is associated with delayed recovery, prolonged length of hospital stay, exorbitant cost, and mortality. The present prospective longitudinal study aimed to evaluate the relationships between the microbial load of trocars used in laparoscopic gynecological surgery, microbiota in surgical sites, and SSI. The final sample consisted of 24 patients, including 68 swab samples and 48 trocars. Microorganisms were recovered in 100.0% of the swabs collected from the umbilicus and vaginal fornix and in 58.3% (14/24) of the swabs collected from skin at the left McBurney's point. Most of the samples collected from trocars (87.5%) did not exhibit bacterial growth, suggesting proper disinfection. In addition, antisepsis was effective for decolonization of the skin to create an aseptic surgical field.