LowGC-type plasmids conferring resistance to sulfonamides have been frequently isolated from manure and manured soil. However, knowledge on the dynamics of plasmid-carrying populations in soil and their response to the presence of sulfonamides is scarce. Here, we investigated effects of the sulfonamide resistance conferring plasmid pHHV216 on the fitness of Acinetobacter baylyi BD413 in soil after application of manure with or without the sulfonamide antibiotic sulfadiazine (SDZ). The persistence of A. baylyi BD413 pHHV216 in competition to its plasmid-free variant was followed in soil microcosms. CFU counts showed a decrease in A. baylyi BD413 in manured soils over the experimental period of 32 days by about 0.5 log units. The proportion of the plasmid-carrying populations decreased from 50 to < 40% in the absence of SDZ, while the proportion of plasmid-carrying BD413 increased from 50 to about 65% with SDZ added. The data suggest that SDZ introduced via manure into soil was bioaccessible, providing a fitness advantage for the plasmid-carrying population of BD413 in soil, while the plasmid conferred a fitness disadvantage when selective pressure by SDZ was absent. In future, this method may be used as a tool for the assessment of bioavailability of antibiotics in soil.