Organic amendments can improve soil fertility and microbial diversity, making agroecosystems more resilient to stress. However, it is uncertain whether organic amendments will enhance the functional capacity of soil microbial communities, thereby mitigating fluctuations in microbial respiration caused by environmental changes. Here, we examined the impacts of long-term organic amendments on the dynamics of microbial catabolic capacity (characterized by enzyme activities and carbon source utilization) and microbial respiration, as well as their interrelationships during a period with fluctuating temperature and rainfall in the field. We then subjected the field soil samples to laboratory heating disturbances to further evaluate the importance of microbial catabolic capacity in explaining patterns of microbial respiration. In both field and laboratory experiments, organic amendments tended to increase the stability of microbial catabolic capacity, but significantly increased the vulnerability of microbial respiration to environmental changes. However, the direction and driving factors of microbial respiration affected by environmental changes differed between the field and laboratory experiments. Environmental changes in the field suppressed the promotional effects of organic amendments on microbial respiration mainly through reducing microbial catabolic capacity, while laboratory heating further enhanced microbial respiration mainly due to increased soil resource availability. Together, these findings suggest that increased microbial respiration variations under organic amendments may potentially increase the uncertainty in predicting soil carbon emissions in the scenario of ongoing climate/anthropogenic changes, and highlight the necessity of linking laboratory studies on environmental changes to field conditions.