Electrospun nanofibers composed of polymers have been extensively researched because of their scientific and technical applications. Commercially available polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHB-HV) copolymers are good choices for such nanofibers. We used a highly integrated method, by adjusting the properties of the spinning solutions, where the cyanophyte Arthrospira (formally Spirulina) was the single source for nanofiber biofunctionalization. We investigated nanofibers using PHB extracted from Spirulina and the bacteria Cupriavidus necator and compared the nanofibers to those made from commercially available PHB and PHB-HV. Our study assessed nanofiber formation and their selected thermal, mechanical, and optical properties. We found that nanofibers produced from Spirulina PHB and biofunctionalized with Spirulina biomass exhibited properties which were equal to or better than nanofibers made with commercially available PHB or PHB-HV. Our methodology is highly promising for nanofiber production and biofunctionalization and can be used in many industrial and life science applications.