Bottas hit the front late on, after a run on Pirelli’s red-marked supersoft tyre - part of a series of qualifying runs - produced a best lap of 1m 23.261s.
That was enough to demote morning pacesetter Lewis Hamilton into second, the Briton having handed over to Mercedes team mate Nico Rosberg in the afternoon.
Rosberg ended up ninth fastest overall, his focus on a full race simulation rather than lap time. He completed 91 laps, to add to Hamilton’s tally of 73 - meaning Mercedes once again led the way in terms of completed miles.
Renault's Kevin Magnussen didn’t improve his morning benchmark and therefore stayed third, clocking up 126 laps in the process.
That tally was bettered by three other drivers - Ferrari's Sebastian Vettel, Red Bull’s Daniel Ricciardo and Toro Rosso’s Carlos Sainz.
Of that trio it was Sainz who completed the most with 166, although he was also the slowest of all three, ending the day seventh overall, fractions behind Ricciardo.
Vettel, meanwhile, was fourth, half a second ahead of McLaren’s Jenson Button - who also broke into triple figures in the lap count on what was a very solid day of work for the Woking team.
But as others racked up the miles, Haas hit issues. Having completed just 23 laps on the opening day, Esteban Gutierrez managed one installation lap and failed to set a time as turbo problems contrived to keep him in the garage throughout.
Sauber also lost time due to technical gremlins in the morning, and while Marcus Ericsson was eventually able to muster 55 laps on his first day in the new C35, he also ran into trouble when he spun into the gravel at Turn 4 after losing his right-rear wheel one hour from the end.
Ericsson ended the day 11th in the timesheets, behind Force India’s Sergio Perez, Rosberg, and the Manor of Pascal Wehrlein.
Wednesday's unofficial testing times from Barcelona
|1||Valtteri Bottas||Williams-Mercedes FW38||1:23.261||108|
|2||Lewis Hamilton||Mercedes F1 W07 Hybrid||1:23.622||0.361||73|
|3||Kevin Magnussen||Renault R.S.16||1:23.933||0.672||126|
|4||Sebastian Vettel||Ferrari SF16-H||1:24.611||1.350||151|
|5||Jenson Button||McLaren-Honda MP4-31||1:25.183||1.922||121|
|6||Daniel Ricciardo||Red Bull Racing-TAG Heuer RB12||1:25.235||1.974||135|
|7||Carlos Sainz||Toro Rosso-Ferrari STR11||1:25.300||2.039||166|
|8||Sergio Perez||Force India-Mercedes VJM09||1:26.036||2.775||128|
|9||Nico Rosberg||Mercedes F1 W07 Hybrid||1:26.298||3.037||91|
|10||Pascal Wehrlein||Manor Racing-Ferrari MRT05||1:27.064||3.803||79|
|11||Marcus Ericsson||Sauber-Ferrari C35||1:27.862||4.601||55|
|12||Esteban Gutierrez||Haas-Ferrari VF-16||-||1|