In this study, we examined 25 actinomycete strains isolated from soil samples collected from different agricultural locations in Baghdad City, Iraq. These isolates were tentatively identified on the basis of their chalky, leathery, waxy, and mucoid colonies. Identifications were confirmed using slide culture techniques and observing the substrate and aerial mycelia and the arrangement of spore chains. Ten of the isolates were established to have antimicrobial-producing activity. To further confirm these isolates as actinomycetes, we performed molecular analyses using 16S rDNA, for which we obtained a characteristic single band of approximately 1500 bp. A selected isolate was studied for its antimicrobial activity against gram-positive and gram-negative bacteria, and we also examined the influence of factors such as carbohydrate sources (glucose, maltose, sucrose, lactose, and starch), and different concentrations of (NH4)2SO4 as a nitrogen source, on antimicrobial production.