Antimicrobial resistance is an increasing global threat, demanding new therapeutic biomolecules against multidrug-resistant bacteria. Antimicrobial peptides (AMPs) are promising candidates for a new generation of antibiotics, but their potential application is still in its infancy, mostly due to limitations associated with large-scale production. The use of recombinant DNA technology for the production of AMPs fused with polymer tags presents the advantage of high-yield production and cost-efficient purification processes at high recovery rates. Owing to their unique properties, we explored the use of an elastin-like recombinamer (ELR) as a fusion partner for the production and isolation of two different AMPs (ABP-CM4 and Synoeca-MP), with an interspacing formic acid cleavage site. Recombinant AMP-ELR proteins were overproduced in and efficiently purified by temperature cycles. The introduction of a formic acid cleavage site allowed the isolation of AMPs, resorting to a two-step methodology involving temperature cycles and a simple size-exclusion purification step. This simple and easy-to-implement purification method was demonstrated to result in high recovery rates of bioactive AMPs. The minimum inhibitory concentration (MIC) of the free AMPs was determined against seven different bacteria of clinical relevance (, , , , and two strains), in accordance with the EUCAST/CLSI antimicrobial susceptibility testing standards. All the bacterial strains (except for ) were demonstrated to be susceptible to ABP-CM4, including a resistant clinical strain. As for Synoeca-MP, although it did not inhibit the growth of or , it was demonstrated to be highly active against the remaining bacteria. The present work provides the basis for the development of an efficient and up-scalable biotechnological platform for the production and purification of active AMPs against clinically relevant bacteria.