Nitrogen (N) and phosphorus (P) availability both control microbial decomposers and litter decomposition. However, these two key nutrients show distinct release patterns from decomposing litter and are unlikely available at the same time in most ecosystems. Little is known about how temporal differences in N and P availability affect decomposers and litter decomposition, which may be particularly critical for tropical rainforests growing on old and nutrient-impoverished soils. Here we used three chemically contrasted leaf litter substrates and cellulose paper as a widely accessible substrate containing no nutrients to test the effects of temporal differences in N and P availability in a microcosm experiment under fully controlled conditions. We measured substrate mass loss, microbial activity (by substrate induced respiration, SIR) as well as microbial community structure (using phospholipid fatty acids, PLFAs) in the litter and the underlying soil throughout the initial stages of decomposition. We generally found a stronger stimulation of substrate mass loss and microbial respiration, especially for cellulose, with simultaneous NP addition compared to a temporally separated N and P addition. However, litter types with a relatively high N to P availability responded more to initial P than N addition and vice versa. A third litter species showed no response to fertilization regardless of the sequence of addition, likely due to strong C limitation. Microbial community structure in the litter was strongly influenced by the fertilization sequence. In particular, the fungi to bacteria ratio increased following N addition alone, a shift that was reversed with complementary P addition. Opposite to the litter layer microorganisms, the soil microbial community structure was more strongly influenced by the identity of the decomposing substrate than by fertilization treatments, reinforcing the idea that C availability can strongly constrain decomposer communities. Collectively, our data support the idea that temporal differences in N and P availability are critical for the activity and the structure of microbial decomposer communities. The interplay of N, P, and substrate-specific C availability will strongly determine how nutrient pulses in the environment will affect microbial heterotrophs and the processes they drive.< Réduire