Successful sod based rotations with row crops have been established at the North Florida Research and Education Center, Quincy, Florida since 2001. On-going research since then has revealed the merits of the sod based system with respect to improved soil physical properties, crop yield, and pest management. A combined knowledge of physical, chemical, biochemical, and microbiological status of the soil is key to understanding the short-term and long-term effects of this system on soil-plant interactions. The present study compares the soil microbiological and biochemical properties in two year Bahia grass-peanut-cotton-winter crop (oats) rotations with conventional peanut-2 yr cotton-winter crop (oats) rotations. Surface soil (0-15cm) cores were composited to obtain a sample representing each cropping sequence. After initial processing, microbial biomass carbon, microbial phosphorus, and enzymes involved in C, P, N, and S cycling were measured in both systems. Both biomass carbon and enzyme activities decreased with depth with higher values at the 0-5 cm level. Biomass carbon was higher (160-300 mg C/kg soil) in the sod based rotations than the conventional rotations (140-170 mg C/kg soil). Activities of beta glucosidase (C cycling), beta glucosaminidase (C and N cycling), acid and alkaline phosphatases (P cycling), and arylsulfatase (S cycling) were all significantly higher in the sod based system. These results suggest that soil microbial biomass and enzyme activities were more favorable in the sod based crop rotations and these, in turn, may be contributing toward the beneficial effects observed in this cropping system.