Both short- and long-term effects of fertilizers on crops and soils are often studied only in arid or paddy soils, whereas less is known about the long-term effects in paddy-upland rotations, particularly with multiple crops and frequent tillage in subtropical areas. Therefore, an 18-year field experiment was initialized to assess the effects of different types of fertilization (no fertilizer; chemical fertilizer (CF); and manure in combination with CF (MCF)) on yield and soil chemical and microbial properties in a crop rotation involving rice (Oryza sativa L., summer), rapeseed (Brassica campestris L., winter), tobacco (Nicotiana tabacum L., the following summer), and hairy vetch (Vicia villosa Roth, the following winter). MCF caused higher yields of rapeseed grains and tobacco leaves than CF after 3 or 4 years of implementing the experiment, while rice yields varied little between MCF and CF, with one exception in 2011. Compared with the initial soil properties, providing soil with MCF increased organic matter (OM), while the opposite trend was found with CF. Higher microbial biomasses, enzyme activities, bacterial operational taxonomic units, and richness and diversity indexes of bacterial communities were found in soils receiving MCF, implying the improvement of soil microbial properties in the paddy-upland rotation system with multiple crops and frequent tillage. The experimental soils under varying fertilization were dominated by four bacterial phyla (Proteobacteria, Acidobacteria, Actinobacteria, and unclassified groups), which accounted for approximately 70% of the 16S rDNA sequences. Among the top 20 predominant bacteria, 14 were commonly found in all soil samples irrespective of which fertilizer treatment was implemented. Thus, the presence of those bacteria was stable in the soil and to some extent was influenced by fertilization. Most of them were facultative anaerobic bacteria, which can adapt to both anaerobic paddy soil and aerobic drylands. The dominant bacteria at various taxonomic levels found in soils might reflect multiple soil processes such as OM turnover, nutrient cycling, physical structure formation, and xenobiotic detoxification.