Liming has been widely used to decrease soil acidity, but its effects on soil nitrogen (N) availability and microbial processes in sugarcane fields are largely unknown. Adjacent sugarcane soils at 26 months after liming (26ML), 14 months after liming (14ML) and with no lime amendment (CK) in Bundaberg, Australia, were selected to investigate the effect of liming on soil N bioavailability and microbial activity in a long-term subtropical sugarcane cropping system. Liming in both 14ML and 26ML treatments significantly increased soil pH (by 1.2–1.4 units) and exchangeable Ca2+ (>2-fold) compared with the CK treatment. The lower concentrations of hot water extractable organic carbon (C) and total N and ammonium-N in the 14ML, compared with the CK and 26ML treatments, can be attributed to the absence of trash blanket placement in the former. Enhanced microbial immobilisation due to improved soil pH by liming (14ML and 26ML treatments) led to increased soil microbial biomass C and N, particularly in the presence of a trash blanket (26 ML treatment), but decreased soil respiration and metabolic quotient indicated that acidic stress conditions were alleviated in the liming treatments. Soil pH was the main factor governing soil enzyme activities, with an overall decrease in all enzyme activities in response to liming. Overall, liming and trash blanket practices improved sugarcane soil fertility. Further study is warranted to investigate the shifts in soil microbial community composition and the diversity and abundance of N-associated functional genes in response to liming in sugarcane fields.