Lysozyme is a potential replacement for antibiotics in the poultry industry. Two trials were conducted using new or used litter to determine the effect of 100 ppm lysozyme in feed on growth performance and intestinal microbiota of broiler chickens in each period of the growth cycle. The new litter trial and used litter trial were each analyzed as a one-way analysis of variance with length of time in which 100 ppm lysozyme was fed to the birds as the main factor (no lysozyme d zero to 35; positive lysozyme d zero to 4; positive lysozyme d 5 to 14; positive lysozyme d 15 to 24; positive lysozyme d 25 to 35; positive lysozyme d zero to 35; positive virginiamycin d zero to 35). Effects of lysozyme were not detected under clean conditions (P > 0.05). When used litter was provided, growth performance was not improved by lysozyme (P > 0.05). However, feeding lysozyme to birds from d 5 to 14 and throughout the trial reduced the number of E. coli in the ileum compared with feeding virginiamycin (positive lysozyme d 5 to 14 and zero to 35, and virginiamycin d zero to 35: 1.7 × 104 cfu g−1, 1.9 × 104 cfu g−1 and 9.3 × 104 cfu g−1, respectively) to birds (P < 0.05). Dietary lysozyme at 100 ppm can change intestinal microbiota of broiler chickens.