The aim of the study was to determine the impact that three cultivation systems—conventional till (CT), reduced till (RT), and strip-till one-pass (ST-OP)—had on the biological parameters of the soil and their relationships with organic matter properties in the row zone (R) and inter-row zone (IR). For this purpose, a long-term static field experiment was carried out, from which soil samples were taken from a depth of 0–20 cm and the following were determined: TOC; TN content and fractional composition of organic matter; activity of dehydrogenases (DEHs), catalase (CAT), alkaline (AlP), and acid phosphatase (AcP); and the abundances of heterotophic bacteria (B), filamentous fungi (F), actinobacteria (Ac), and cellulolytic microorganisms (Ce). Soil samples for biological parameter tests were collected in summer (July) and autumn (October). RT and ST-OP increase the content of TOC, TN, carbon, and nitrogen in the humic and fulvic acid fractions. For the studied groups of microorganisms, the conditions for development were least favourable under CT cultivation. The results show that in July, the activities of DEH and CAT were the highest in ST-OP, whereas in October, they were the highest under CT. AlP and AcP activity were markedly the highest under ST-OP in both months. Enzyme activity was significantly the highest in the IR zone. The results indicate that, of the calculated multiparametric indicators, (AlP/AcP, GMea, BIF, BA12, and TEI), BA12 is a sensitive biological indicator of soil quality.