Ten-week-old Hy-Line Commercial W-36 pullets were spray-vaccinated with MYCOVAC-L® at the manufacturer's recommended dosage (1×) or at 15 times that rate (15×). At 22 or 45 wk of age, subsets of 1×- and 15×-vaccinated pullets were challenged with the virulent Mycoplasma gallisepticum (MG) strain R_low. Percent hen-day egg production was determined through week 55. Analyses for treatment effects on overall (22–56 wk) percent hen-day egg production revealed no significant differences between nonchallenged 1× and nonchallenged 15× MYCOVAC-L® treatments. Among 1× MYCOVAC-L®–vaccinated groups, R_low challenge at 45 wk corresponded to significantly (P ≤ 0.01) lower overall egg production compared with the unchallenged 1×-vaccinated control (70.88% vs. 79.15%, respectively). Conversely, at the 15× MYCOVAC-L® dosage level, overall egg production was not significantly affected by virulent MG challenge at 45 wk compared with its unchallenged counterpart (84.09% vs. 81.03%, respectively) and could indicate increased protection from virulent MG challenge. Serologic monitoring indicated the virulent MG challenge was consistently (100%) associated with seroconversion. Comparisons among the nonchallenged experimental treatments indicated that vaccinations at the 15× MYCOVAC-L® dosage rate were associated with a greater seroconversion rate at weeks 21, 27, and 44, but not at week 50.