Microbial interactions play a critical role in ecosystem stability. However, the degree of competition and cooperation that are reflected in the observed composition of microbial communities and their response to alterations in nutrient availability has not been systematically investigated. Thus, we utilized monocultures and pairwise co-cultures in order to measure changes in the growth, niche and interactions among sixteen cultured rhizosphere soil bacteria across different carbon types and nutrient availabilities. Our analysis showed that interactions differed among strain pairs and across carbon sources. We further found that resource limitation resulted in ∼3% greater increases in microbial inhibition and more negative interactions. In contrast, high nutrient concentrations allowed for bacterial metabolic niche expansion, a decrease in inhibition (79.69% of cases) and stronger facilitation. Overall, our results show that the prevalence of cooperation is higher in nutrient-rich conditions, thus revealing the critical role that resource availability plays in shaping microbial interactions.