One of the greatest contemporary challenges in terrestrial ecology is to determine the impact of climate change on the world's ecosystems. Here we investigated how wetting patterns (frequency and intensity) and nutrient additions altered microbial biomass and CO2-C loss from a semi-arid soil. South-western Australia is predicted to experience declining annual rainfall but increased frequency of summer rainfall events when soil is fallow. Agricultural soils (0-10 cm at 10 °C or 25 °C) received the same total amount of water (15 mL over 30 days) applied at different frequency; with either nil or added nitrogen and phosphorus. Smaller more frequent wetting applications resulted in less CO2-C loss (P < 0.001); with cumulative CO2-C loss 35% lower than a single wetting event. This coincided with increased microbial biomass C at 25 °C but a decline at 10 °C. Increasing nutrient availability decreased CO2-C loss only under a single larger wetting event. While bacterial and fungal abundance remained unchanged, archaeal abundance and laccase-like copper monooxidase gene abundance increased with more frequent wetting at 25 °C. Our findings suggest smaller more frequent summer rainfall may decrease CO2 emissions compared to infrequent larger events; and enhance microbial C use efficiency where sufficient background soil organic matter and nutrients are available.