In Mississippi, spent poultry litter is used as fertilizer. Nutrient and bacterial levels in litter and nutrient levels in litter-fertilized (L+) soil are known, but less is known of bacterial levels in L+ soil. This study compared contiguous L+ and non-litter-fertilized (L-) soils comprising 15 soil types on five farms in April through May 2009. Levels of pH; NO-N; and Mehlich-3-extractable (M3) and water-extractable (WE) P, Ca, K, and Cu were higher in L+ than in L- soil. Total C; total N; NH-N; and M3 and WE Na, Fe, and Zn did not differ in L+ and L- soil. Bacterial levels were higher in 0- to 5-cm than in 5- to 10-cm cores. Levels were higher in L+ than in L- soil for culturally determined heterotrophic plate counts and staphylococci and were lower for total bacteria estimated by quantitative polymerase chain reaction (qPCR) of 16S rRNA, but cultural levels of thermotolerant coliforms, , , and enterococci were not different. Cultural presence/absence (CPA) tests and qPCR for spp., spp., and spp. detected only spp., which did not differ in L+ (CPA = 77% positive samples; mean qPCR = 0.65 log genomic units [gu] g) and L- (CPA = 70% positive samples; mean qPCR = 0 log gu g) soils. Litter applications were associated with higher levels of pH, P, Cu, heterotrophic plate counts, and staphylococci. Fecal indicator and enteric pathogen levels were not affected. We conclude that, although some litter-derived nutrients and bacteria persisted between growing seasons in L+ soils, enteric pathogens did not.