As two commonly-used non-selective herbicides, glyphosate (GP) and diquat (DQ) are easily co-resided in lateritic paddy soil due to the rich iron/aluminum oxides, nevertheless there is limited information on their co-impact on microbial diversity and community structure in this type soil. In this study, the short-term effects of combined GP and DQ on soil microbial diversity and community structure shifts were investigated in lateritic paddy soil from a tropical agricultural region (Hainan, China) based on 16S rRNA and ITS high-throughput sequencing technology. The results showed that mixed herbicides promoted the abundance of Streptomyces in bacteria (0.45-1.84%) and Curvularia in fungi (0.01-5.85%), while GP and DQ had inhibitory effects on the abundance of Streptomyces (0.13-2.21%) and Curvularia (0.03-1.13%), which were significantly different with their single exposure (p < 0.05); the combined application of the two herbicides aggravate the adverse effect on the diversity of soil fungal community (p < 0.05), although their mixture did not have a greater impact on the soil bacteria abundance/diversity and fungi abundance (p > 0.05). Results suggested that the combined application of GP and DQ affected the fungal diversity although they did not cause other significant negative effects on soil microorganisms, hinting that more attention should be paid to the mixed effect caused by GP and DQ on specific fungal populations in lateritic paddy soil.