Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis or Mycobacterium bovis and still remains one of the world’s biggest global health burdens. Recently, engineered polyhydroxyalkanoate (PHA) biobeads produced in both E. coli and Lactococcus lactis displaying mycobacterial antigens were found to induce significant cell mediated immune responses in mice. We observed that such PHA beads suspensions contained co-purifying host cell proteins that we hypothesise have the potential to induce immunity. In this study we aimed to develop PHA beads produced in mycobacteria (mycobacterial PHA biobeads, MBB) and test their potential as TB vaccine in a mouse model. As a model organism, nonpathogenic Mycobacterium smegmatis was engineered to produce MBB or MBB displaying mycobacterial antigens Ag85A and ESAT-6 on their surface (A:E-MBB). These MBB were used in a mouse vaccination and TB challenge trial in comparison with BCG vaccination. Lymphocytes from mice vaccinated with MBB showed IFN-γ and IL-17 responses when restimulated in vitro with MBB. Using flow cytometric analysis we found induction of IFN-γ and IL-17 production from non-classical CD3 T cells (CD4 and CD8 negative) in cells from mice receiving MBB. This contrasts with the response from the same cells from mice that received BCG vaccination, where this cytokine response was low. Also, ex vivo IFN-γ production was evident in a subset of granulocytes from all mice including unvaccinated animals but more granulocytes from MBB immunised mice produced IFN-γ than from unvaccinated or BCG immunised mice. After TB challenge, lung and spleen cfu counts suggested that 4 out of 5 BCG vaccinated and 2 out of 5 MBB-A:E vaccinated mice were protected. This study proves the feasibility of production of biobeads in mycobacteria and also indicates their potential efficacy as vaccines against tuberculosis.