Two independent experiments were performed to evaluate the effect of nisin alone or with monensin on gut microbiota, gut microbial activities, and histomorphology (exp 1) and the effect of nisin application in a dose‒response manner on the growth performance of broiler chickens (exp 2). A total of 900 one-day-old female Ross 308 chicks (400, exp 1; 500, exp 2) were randomly distributed to four groups (exp 1; 10 replicate pens per treatment with 10 birds each), i.e., NA, no additives; MON, monensin (100 ppm); NIS, nisin (2700 IU/kg diet); and MON + NIS, a mixture of monensin (100 ppm) and nisin (2700 IU/kg diet); or 5 treatments (exp 2), NA, no additives; NIS100, nisin (100 IU/kg diet); NIS200, nisin (200 IU/kg diet); NIS400, nisin (400 IU/kg diet); and NIS800, nisin (800 IU/kg diet). Nisin supplementation positively affected the microbiota of the gut by reducing potentially pathogenic bacterial populations in the jejunum and ceca. The bacterial fermentation in the jejunum was significantly lowered by nisin addition. The addition of nisin from 100 IU to 800 IU decreased the FCR value over the entire experimental period. According to the results, nisin can be considered a natural dietary supplement for broiler chickens.