Rice may have negative effects on the soil due to intensive levelling tasks required for flood irrigation. Therefore, rotations including rice influence soil physical, chemical and biological properties differently. In our study, the effects of rotations with rice on biological properties were evaluated and associated with the capacity of the soil to supply N to crops. Furthermore, the relationships among the variables were studied and those most sensitive to detect the resulting changes were determined. The study was conducted on four crop sequences over a 4-year period: rice monoculture (RR), rice–soybean (RS), rice–soybean–maize–soybean (RSMS) and rice–pasture (RP). The four rotations evaluated had a strong effect on soil properties. Principal components analysis showed that RR and the RP rotation were discriminated clearly, while RSMS and RS were in the middle of the biplot, forming two different groups. Microbial biomass N (MBN), potential of N mineralization measured by anaerobic incubations (PMN-AI) and the microbial biomass C to N ratio were the variables that differed most the studied rotations. The PMN-AI variable was positively associated with MBN, microbial biomass C, organic C, total N, urease, and fluorescein diacetate hydrolysis (FDA) variables. The potential of N mineralization measured with hot KCl neither evidenced differences among the evaluated situations nor was associated to the other variables. This is why it is assumed this is not a good soil quality indicator. The differences found in microbial analysis indicate that microbiological variables (MBC, MBN), nitrogen availability index (PNM-AI), and biochemical variables (FDA) were sensitive variables to evaluate soil rotations’ effects and they might be used as good soil quality indicators once their critical values have been determined for different conditions.