The aim of the study was to compare the effect of conventional, simplified, and organic farming systems on changes in the content of soil organic carbon, organic matter fractions, total nitrogen, and the enzymatic activity. The research was conducted from 2016–2018 on arable land in the south-eastern part of Poland. The selected soils were cultivated in conventional tillage (C_Ts), simplified tillage (S_Ts), and organic farming (O_Fs) systems. The analyses were performed in soil from the soil surface layers (up to 25 cm depth) of the experimental plots. The highest mean contents of soil organic carbon, total nitrogen, and organic matter fractions were determined in soils subjected to the simplified tillage system throughout the experimental period. During the study period, organic carbon concentration on surface soil layers under simplified tillage systems was 31 and 127% higher than the soil under conventional tillage systems and organic farming systems, respectively. Also, the total nitrogen concentration in those soils was more than 40% and 120% higher than conventional tillage systems and organic farming systems, respectively. Moreover, these soils were characterised by a progressive decline in SOC and Nt resources over the study years. There was no significant effect of the analysed tillage systems on the C:N ratio. The tillage systems induced significant differences in the activity of the analysed soil enzymes, i.e., dehydrogenase (DH) and catalase (CAT). The highest DH activity throughout the experiment was recorded in the O_Fs soils, and the mean value of this parameter was in the range of 6.01–6.11 μmol TPF·kg−1·h−1. There were no significant differences in the CAT values between the variants of the experiment. The results confirm that, regardless of other treatments, such as the use of organic fertilisers, tillage has a negative impact on the content of SOC and organic matter fractions in the O_Fs system. All simplifications in tillage reducing the interference with the soil surface layer and the use of organic fertilisers contribute to improvement of soil properties and enhancement of biological activity, which helps to maintain its productivity and fertility.