The effects of using effluent bacteria (EB) and solid- (SAB) and liquid- (LAB) associated bacteria and diaminopimelic acid (DAPA) or purine bases (PB) and partially substituting alfalfa hay (AH) by a concentrate including olive cake on the microbial N flow (MNF) and amino acids (AA) flow were investigated with continuous culture fermenters fed AH and a mixture of AH and a concentrate containing barley grains and two-stage olive cake (2:1 ratio) without (AHCO) or with polyethylene glycol (PEG) (AHCOP). The MNF was not different among diets with SAB or LAB (p = 0.302 and 0.203, respectively) and DAPA, but differed with PB (p = 0.021 and 0.014, respectively). With EB both markers detected similar differences, AHCOP showing a higher value (p < 0.05) than AH and AHCO. The MNF was higher (p < 0.001) with PB than DAPA. Daily flow of non-essential AA was not different (p = 0.356) among diets but essential AA flow was higher (p < 0.05) for AH and AHCOP than for AHCO. The SAB presented lower (p < 0.05) total AA than LAB and higher total AA (p < 0.05) for diet AH than AHCO. The AA profile of EB was similar to that of LAB, but alanine and leucine were higher (p < 0.05) in EB than in LAB. Microbial contribution to AA flow was 45.4%, 55.6% and 58.1% for diets AH, AHCO and AHCOP respectively. With both markers, microbial AA flow was higher (p < 0.05) for diet AHCOP compared with AH (451 and 355 mg/day, respectively), but not different (p > 0.05) for AHCOP and AHCO (389 mg/day). The results would indicate that olive cake could be used in the practical feeding of small ruminants without negatively affecting microbial AA N supply.