In 2019, 71 years after the establishment of a static fertiliser experiment, the chemical and biological properties of Luvisol soil with sandy-loam grain-size composition were determined. Soil samples were taken from six fertilised treatments: half-dose nitrogen, phosphorus and potassium in mineral fertilisers (½ NPK); full-dose nitrogen, phosphorus, potassium (NPK); manure fertilisation + nitrogen, phosphorus, potassium, magnesium and liming (FYM NPK Mg Ca); manure + mineral fertilisers without magnesium and liming (FYM NPK); manure + nitrogen and phosphorus (FYM NP); manure + nitrogen and potassium (FYM NK). The soil was tested in two layers at depths of 0–20 cm and 20–40 cm. Soil samples were tested for: pH in 1 M KCl (pH); electrical conductivity (EC); organic carbon content (OC); content of available phosphorus (Pa), potassium (Ka), magnesium (Mga) and sulphate sulphur (S-SO4); total number of bacteria (Bt), cellulolytic microorganisms (Bc), fungi (Ff) and actinomycetes (Ac); and alkaline phosphatase (AlP), acid phosphatase (AcP) and arylsulphatase (ArS) activity. The fertilisation that most favourably affected the chemical and biological properties of the soil was FYM NPK Mg Ca. This fertilisation increased: pH and EC; OC, Ka and Mga contents; Bt and Bc abundance; and AlP activity relative to all the methods of mineral and organic–mineral fertilisation that did not include all the ingredients of mineral fertilisers. On the other hand, the least favourable soil properties were formed by ½ NPK fertilisation in the 0–20 cm layer, and by the long-term use of mineral fertilisers only in the 20–40 cm layer.