This research investigated the effect of microwave irradiation (MWI) on cell disintegration in municipal secondary sludge (MSS). A representative MSS Gram-positive bacterium (Bacillus subtilis) and Gram-negative bacteria (Acinetobacter calcoaceticus and Pseudomonas aeruginosa) were pure cultured separately and treated using MWI. Compared to untreated controls, MWI significantly increased the soluble chemical oxygen demand (COD) (1.8-4.0-fold), soluble protein concentration (1.1-1.8-fold), and soluble carbohydrate concentration (3.2-14.1-fold), with greater increase in the Gram-negative bacteria. After MSS was MWI-treated with different irradiation times, from 0 to 9 min, soluble COD increased gradually from 0.14 to 2.38 g/L (i.e., 72-fold). Effective disintegration of Gram-negative cell walls and of MSS by MWI was confirmed by scanning electron microscopy. These findings suggest that MWI could be an effective pretreatment method for MSS that is dominated by Gram-negative microorganisms.