The changes of physicochemical and biochemical parameters of a silty loam (S1) and sandy loam (S2) vineyard soils added with spent mushroom substrate (SMS) or SMS composted with ophite (OF) as rock dust (SMS + OF) were studied. Two doses of SMS or SMS + OF (25 and 100 Mg ha−1) were applied for two consecutive years (2020–2021) and changes of soil physicochemical parameters, and dehydrogenase activity (DHA), respiration (RES), microbial biomass (BIO), and the phospholipid fatty acids (PLFAs) profile were assayed on a temporal basis. The results showed an increase in soil organic carbon (OC) content, total and mineralised N, P, and K, especially when the highest SMS dose was applied to soils. Repeated application caused OC content over time up to 2.3 times higher than initial content in the silty loam soil. This increase was not observed in sandy soil, possibly due to a higher bioavailability of OC, as indicated by the evolution of extractable humic acid/fulvic acid pools. In both soils, all biochemical parameters increased after amendment, being favoured both by the OC and by the presence of OF. Significant positive correlations were found between DHA, RES and BIO, and OC content especially in the first part and then levelled off after the second dose application. Total bacterial or fungal PLFAs patterns reflected the variation of BIO by SMS application. The higher growth of fungi vs. bacterial community in amended soils was recorded after the first SMS application, although the opposite effect occurred after the second application, with similar results in both soils. The findings indicate that the application of SMS or SMS + OF in vineyard soils could be an appropriate agronomic management practice for maintaining soil sustainability, although doses and application times of these amendments should first be evaluated depending on soil texture.