The present study investigates the effects of the preslaughter fasting on pH and bacterial contamination in Akkaraman and Anatolian Merino sheep. Included in the study were 10 Akkaraman and 10 Anatolian Merino sheep (n = 20) that were fasted for 24 h prior to slaughter, and a further 10 Akkaraman and 10 Anatolian Merino sheep (n = 20) that continued to be fed until slaughter as a control group. The total mesophilic aerobic bacteria (TMAB) measured at 0 and 24 h in the fasting Akkaraman and Anatolian Merino sheep were 4.87/4.79 and 4.75/5.57 log CFU/g, respectively, and the corresponding Enterobacteriaceae counts (TEC) were 3.37/2.38 and 3.20/1.99 log CFU/g, respectively. Overall, both contamination values increased during the fasting period. The effects on TMAB counts (5.22 ± 0.624 log CFU/g and 4.77 ± 0.408 log CFU/g) were found to be statistically significant, and a significant interaction effect was also observed. While fasting did not have a statistically significant effect in the Akkaraman breed, it had a significant effect in Anatolian Merino sheep (p < 0.01). The pH values of Akkaraman and Anatolian Merino sheep, both fasted and nonfasted, were measured immediately after slaughter (pH0) and at 24 h postslaughter (pH24). The mean pH0 values were 7.55 ± 0.162 and 7.45 ± 0.203, respectively, whereas the pH24 values were 5.72 ± 0.123 and 5.78 ± 0.141, respectively. No statistically significant differences were observed between groups. In conclusion, preslaughter fasting reduces intestinal content and, consequently, bacterial load, indicating a positive effect on microbial contamination. Furthermore, the absence of significant differences in pH values at 0 and 24 h postslaughter suggests that fasting does not adversely affect animal handling conditions prior to slaughter or the overall quality of the meat.