A novel method to exfoliate the montmorillonite clay was developed previously to generate random nanosilicate platelets (NSP), one kind of delaminated clay. To improve their dispersion in a polymer, we modified NSPs by three types of surfactants (cationic Qa, nonionic Qb, and anionic Qc) in this study and used them to prepare nanocomposites with polyurethane (PU). The zeta potential, antimicrobial ability, and biocompatibility of these surfactant-modified NSPs (abbreviated "NSQ") were characterized. It was found that the zeta potential of Qa-modified NSP (NSQa) was positive, whereas those of NSP and the other two NSQs (NSQb and NSQc) were negative. All NSQ presented less cytotoxicity than NSP. NSQa and NSQc showed excellent antimicrobial activities against S. aureus (Gram-positive strain) and E. coli (Gram-negative strain). The nanocomposites of NSQ with PU were then characterized for surface and mechanical properties, cell attachment and proliferation, antimicrobial activity in vitro, and biocompatibility in vivo. A higher surfactant to NSP ratio was found to improve the dispersion of NSQ in PU matrix. The mechanical properties of all PU/NSQ nanocomposites were significantly enhanced. Among various NSQ, only NSQa were observed to migrate to the composite surface. The attachment and proliferation of endothelial cells and fibroblasts in vitro as well as biocompatibility in vivo were significantly better for PU/NSQa containing 1% of NSQa than other materials. The microbiostasis ratios of PU/NSQ nanocomposites containing 1% NSQa or NSQc were >90%. These results proposed the safety and potential antimicrobial applications of surfactant-modified delaminated clays and their nanocomposites with PU polymer.

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