Research was carried out on the impact of long-term use of cattle manure (30 t ha−1 FYM) and various doses of N (0, 40, 60 and 120 t ha−1) in the form of ammonium nitrate on the following soil parameters: salinity, hydrolytic acidity, total exchangeable base cations, cation exchange capacity, degree of base saturation of the sorption complex, total organic carbon and total nitrogen content, dissolved organic matter, fractional composition of organic matter and content of bioavailable macroelements: phosphorus, potassium and magnesium in the soil of a multi-year static field experiment. The activities of dehydrogenases, catalase, alkaline and acid phosphatase and proteases were also tested. A significant effect of FYM and N fertilisation on the content of bioavailable macroelements was found. The application of manure at a dose of 30 t ha−1 mitigated the negative effects of the application of N at a dose of 120 kg ha−1. A higher content of total organic carbon (8.42 g kg−1) and humic acid fraction (1761 mg kg−1) and higher values of the CHA/CFA ratio (0.79; parameters that are indicators of soil quality) were found in the soil fertilised with manure compared to the soil without manure added (TOC—7.00 g kg−1; CHAs—1285 mg kg−1; CHAs/CFAs 0.66). The activity of the tested enzymes was also significantly determined by the applied fertilisation. Enzyme activity was highest in the soil to which manure had been applied. Nitrogen fertilisation varied in its impact on the activity of enzymes according to the specifics of each enzyme. The content of humic acids and CHA/CFA values correlated positively with the content of soil minerals and the activity of dehydrogenases, catalase, alkaline and acid phosphatase and GMea and TEI indices. Dehydrogenases and acid phosphatase can be considered enzymes that take part in transforming organic matter towards the formation of FAs.