Cover cropping is a well-established strategy to improve soil health, especially in arid and semi-arid agricultural systems. Benefits to soil health are often mediated via effects of cover crops on soil microbial community structure, function and diversity, crucial for regulating soil biogeochemical cycles, eventually promoting agricultural sustainability. However, limited water availability is a major constraint for both cover crop growth and soil microbial activity. This study sought to characterize and elucidate the shifts in soil microbial community structure in response to different cover crops and differential irrigation treatments using phospholipid fatty acid (PLFA) profiling in southern New Mexico. We tested five cover crop treatments: Pisum sativum (Australian winter pea), Hordeum vulgare cv. Stockford (barley), Brassica juncea cv. Caliente 199 (brown mustard), a three-way mix, and a fallow control — in combination with irrigation treatments of one, two, or three irrigation applications — in a split-plot design over two years. Zea mays (sweet corn) was grown as the summer cash crop. We collected soil samples just after cover crop planting in the fall of 2018, following second year cover crop termination but during Z. mays growth in June 2020, and after the second season of Z. mays growth in October 2020. Differential irrigation treatments did not lead to consistent patterns of change under any cover crop or irrigation treatment. However, PLFA parameters in cover cropped compared to winter fallow plots tended to decrease under one and three irrigations but increased with two irrigations. Changes were more common for bacterial than for fungal PLFA biomarkers, and more common in B. juncea and H. vulgare cover crops than in P. sativa or the mix. It is important to note that, while cover crop effects were inconsistent, cover cropping did lead to some shifts in PLFA biomarkers, even in the short two-year period of cover cropping.