Immobilizing antimicrobial substances onto biocompatible materials is an important approach for the design of novel, functionalized medical devices. By choosing antimicrobial substances from innate immune systems, the risk for development of resistance in pathogenic microbes is lower than if conventional antibiotics are used. Combining natural antimicrobial peptides and bactericidal enzymes with strong and elastic spider silk through recombinant protein technology would enable large-scale production of materials that could serve as functionalized wound dressings. Herein, fusion proteins with the engineered spider silk sequence 4RepCT and five different antimicrobial substances were constructed using two different strategies. In the first, the fusion proteins had a His-tag as well as a solubility-enhancing domain N-terminally to the antimicrobial agent during expression. The tags were cleaved and separated from the target protein during the purification process. The other approach provided a His-tag but no additional solubility domain. The antimicrobial agents included in the work were a charge engineered enzyme and four antimicrobial peptides herein called Peptide A, Peptide B, Peptide C and Peptide D. Four out of five fusion proteins could be expressed in Escherichia coli without exhibiting noticeable toxicity to the host. However, most target proteins were found in the non-soluble fraction. For D-4RepCT, neither soluble nor non-soluble proteins were identified. An operating strategy for expression and purification of antimicrobial spider silk proteins was developed, where the construct system providing the solubility-enhancing domain N-terminally to the antimicrobial sequence, and long time expression at low temperatures is a promising approach. The fusion proteins A-4RepCT and C-4RepCT could be produced in adequate amounts, and they proved to possess the ability to assemble into stable fibers. When incubating solutions of Escherichia coli on the functionalized silk material A-4RepCT, it showed to decrease the number of living bacteria in solution, in contrary to wild-type 4RepCT on which bacteria continued to proliferate. Initial studies of the viability of bacteria adhered to the surface of the functionalized spider silk are so far inconclusive. A larger sample size, complementary experiments and methodology optimization is needed for a proper assessment of antibacterial properties. However, preliminary results for the development of antimicrobial spider silk are positive, and the approach elaborated in this work is believed to be applicable for the construction of functional spider silk with a wide range of natural antimicrobial agents for future wound healing applications.