Land application of swine lagoon effluent (SLE) often changes soil pH and the concentrations of nutrients and trace metals. We hypothesize that fertilization-associated alteration in soil properties would have long-lasting effects on soil microbial community and the effects would depend on fertilization rates. In this study, microbial community composition and structure were characterized with phospholipid fatty acid (PLFA) profiles. Soils (0-7.5 cm depth) were sampled in March 2004 from the field plots planted with bermudagrass and tall fescue that received 0-600 kg plant available N (PAN) ha- superscript 1(B year- superscript 1(B of SLE or ammonium nitrate (AN) from 1999 to 2001. We observed that PLFA profiles differed significantly (P < 0.05) between soils supplied with SLE versus AN, specifically at the highest application rate. PLFA profiles were strongly correlated with fertilization-associated changes in soil pH (Pearson’s correlation coefficient r = 0.80, P < 0.01) and Mehlich III extractable Ca (r = 0.78, P < 0.01) and Mg (r = 0.72, P < 0.01). In soils receiving 200 and 400 kg PAN ha- superscript 1(B year- superscript 1(B of SLE or AN, microbial community was enriched slightly with fungi (P < 0.01). In soils receiving 600 kg PAN ha- superscript 1(B year- superscript 1(B of SLE or AN, however, microbial community was stressed as evidenced by the reduction in microbial monounsaturated fatty acids and by the increase in the ratios of saturated-to-unsaturated fatty acids and of cyclopropyl fatty acids-to-their monoenoic precursors. These changes in PLFA biomarkers for stress were significantly (P < 0.01) correlated with soil pH and Mehlich III extractable Ca and Mg, but not with soil properties such as total soil C, soluble soil organic C, soil inorganic N or Mehlich III extractable soil P. Our results indicated that soil microbial community was negatively affected when soils received SLE at an application rate of 600 kg PAN ha- superscript 1(B year- superscript 1(B in this forage production system.